Purification of GST-fusion - GST-fusion protein: How to express and purify (Feb/09/2007 )

I have taken ~30 amino acids of a protein and fused it to GST in pGEX 2T and want to use it as a kinase substrate. Does anyone know how to express this thing in bacteria (they are already in DH5 alpha cells) and purify it?

ThanksDOCDTT

-DOCDTT-

Use Ni-Sepharose column for purification. Use thrombin enzyme to cleave off your GST.

Check out Amersham Bioscience for more details about GST system. You can get free download from here.

I hate to be pedantic, but you'd be better served by using a Glutathione (GSH)-Sepharose, rather than Ni. Ni is for His tag systems, not GST tag

-swanny-

And you'd be better off using BL21s than DH5s.

-jaknight-

QUOTE (jaknight @ Feb 11 2007, 05:46 AM)

And you'd be better off using BL21s than DH5s.

Hi all,Thanks for the replies.

I'll be certain to use GSH-sepharose - I hope I know that much. As for the cells, I guess it's BL21. The post-doc in my lab says DH5 - is she absolutely wrong or are BL21's just better? If she is, then I am having major doubts about her...

DOCDTT

-DOCDTT-

BL21 is an expression strain. It gives better protein yields but could mess up your plasmid. DH5 is a cloning strain. Plasmid integerity is better maintained but protein yeild would probably be less.

It not absolutely wrong. One could use DH5 cells but BL21 would probably serve you better

-perneseblue-

QUOTE (perneseblue @ Feb 11 2007, 02:02 PM)

BL21 is an expression strain. It gives better protein yields but could mess up your plasmid. DH5 is a cloning strain. Plasmid integerity is better maintained but protein yeild would probably be less.

It not absolutely wrong. One could use DH5 cells but BL21 would probably serve you better

That's the ticket...Thanks perneseblue

-DOCDTT-

BL21 has some ohm- gene (protease encoding gene) i think. That's why it will be better choice for expression. Am I right? Just want to make sure.